Accumulation of bile acids within the hepatocyte is thought to play a key role in liver injury during cholestasis. Although the initial insult in certain hepatobiliary diseases such as primary biliary cirrhosis may be immunological, cell injury is probably exacerbated by direct chemical damage from the hydrophobic bile acids. Although the cytotoxicity of hydrophobic bile acids to hepatocytes and a variety of other cell types has been attributed to the membrane disruptive effects from their detergent properties, it is now apparent that nondetergent mechanisms are also involved. In contrast, hydrophilic bile acids such as ursodeoxycholic acid (UDCA) and its taurine and glycine conjugates appear to protect against cholestasis and the toxicity induced by the hydrophobic bile acids (Heuman et al., Gastroenterology, 100, 203-211 (1991) and Heuman et al., Gastroenterology, 106, 1333-1341(1994)). Although the mechanism of action is not entirely understood, the oral administration of UDCA markedly improves clinical and biochemical indices in some chronic liver diseases (Podda et al., Gastroenterology, 98, 1044-1050 (1990); Chazouilleres et al., J. Hepatology, 11, 120-123 (1990); and Poupon et al., N. Engl. J. Med., 330, 1342-1347 (1994)). This protective effect appears to result from mechanisms beyond simply displacing toxic bile acids from the liver.
Bile acid-induced toxicity is typically characterized by hepatocyte swelling, disruption of membrane plasma integrity, and release of intracellular constituents. As a consequence, liver cell death has been characterized as loss of hepatocellular function associated with necrosis. Widespread hepatocyte necrosis, however, is not a prominent feature in most cholestatic liver diseases. In fact, it now appears that hepatocyte cell death occurs more commonly by apoptosis than necrosis (Columbano et al., J. Cell. Biochem., 58, 181-190 (1995)). Apoptosis, or programmed cell death, is characterized by distinctive morphologic and biochemical changes including cell shrinkage, loss of intercellular membrane contact, progressive condensation of chromatin and cytoplasm, and subsequent nuclear fragmentation. These events culminate in the characteristic formation of apoptotic bodies, consisting of nuclear fragments and intact cell organelles surrounded by plasma membrane. The internucleosomal degradation of DNA, which results in fragmentation in multiples of 180 base pairs, and the consequent appearance of a characteristic DNA ladder by gel electrophoresis has become an identifying feature of apoptosis at the molecular level.
Hydrophobic bile salts such as glycodeoxycholate and glycochenodeoxycholate directly induce apoptosis in isolated rat hepatocytes (Spivey et al., J. Clin. Invest. 92 17-24 (1993) and Patel et al., J. Clin. Invest., 94, 2183-2192 (1994)). Moreover, it has been reported that bile salt induced apoptosis of hepatocytes involves activation of the protease cathepsin B through the protein kinase C-dependent pathway (Jones et al., Am. J. Physiol., 272 G1109-G1115 (1997)). Features of apoptosis have been observed in several types of liver diseases. In fact, it was recently reported that nuclear DNA fragmentation and de novo Bcl-1-2 expression were increased in primary biliary cirrhosis, and significantly inhibited in patients treated with UDCA (Koga et al., Hepatology, 25, 1077-1084 (1997)). Although the precise molecular mechanism of cytoprotection by UDCA is not completely known, it has been shown that ursodeoxycholate reduces the mitochondrial membrane damage from certain hydrophobic bile acids (Botla et al., J. Pharmacol. Exp. Ther., 272, 930-938 (1995)). In fact, the results suggested a physiochemical explanation for the bioenergetic form of cell injury associated with hydrophobic bile salts. UDCA cytoprotection may, in part, be due to inhibition of bile salt-induced mitochondrial membrane permeability. It is now apparent that disruption of mitochondrial function is a key factor in the genesis of apoptosis (Reed et al., Nature (Lond.)., 387, 773-776 (1997)). This is supported by the observation that the cell nucleus and DNA fragmentation may not be required for cells to undergo apoptosis.
There are a number of agents other than hydrophobic bile acids that induce apoptosis. Furthermore, there are a number of mechanisms by which apoptosis is induced. Examples of such agents include TGF-xcex21, anti-Fas antibody, okadaic acid, and ethanol. Thus, there is a need for agents that are inhibitory to such inducers of apoptosis which are unrelated to hydrophobic bile acids.
The present invention provides a method for limiting apoptosis (i.e., programmed cell death) of a mammalian cell population. The method comprises contacting the cell population with an effective amount of ursodeoxycholic acid, a salt thereof, an analog thereof, or a combination thereof, wherein the apoptosis is induced by a nonmembrane damaging agent, such as TGF-xcex21, anti-Fas antibody, or okadaic acid. The cell population can include, for example, hepatocytes and astrocytes. The contacting step can be performed in vitro, in vivo, and a combination thereof. As used herein, xe2x80x9cin vitroxe2x80x9d is to be distinguished from xe2x80x9cin vivo.xe2x80x9d In vitro refers to an artificial environment location of the cell population to be treated, such as a cell culture in a tissue culture dish. In vivo refers to a natural environment location of the cell population to be treated, such as in a mammalian body. Preferably, the cell population is a human cell population, and the contacting step involves administering an effective amount of ursodeoxycholic acid, a salt thereof, an analog thereof, or a combination thereof.
One aspect of the present invention provides a method that includes a step of administering to a patient an effective amount of ursodeoxycholic acid, a salt thereof, an analog thereof (e.g., glyco- and tauro-), or a combination thereof. Preferably, the step of administering comprises administering parenterally or intravenously.
The present invention also provides a method for limiting apoptosis of a mammalian cell population, the method comprising contacting the cell population with an effective amount of ursodeoxycholic acid, a salt thereof, an analog thereof, or a combination thereof, wherein the apoptosis is induced by ethanol.
Another aspect of the present invention is a method for limiting apoptosis of a human cell population. Preferably, the method includes contacting the cell population with an effective amount of a hydrophilic bile acid, a salt thereof, an analog thereof, or a combination thereof, wherein the apoptosis is induced by a hydrophobic bile acid.
Yet another aspect of the invention is a method for limiting apoptosis of a mammalian cell population, wherein the method includes contacting the cell population with an effective amount of a hydrophilic bile acid, a salt thereof, an analog thereof, or a combination thereof, wherein the apoptosis is induced by TGF-xcex21, anti-Fas antibody, or okadaic acid.
Still another aspect of the present invention is a method for inhibiting apoptosis associated with a nonliver disease in vivo, the method including administering ursodeoxycholic acid, a salt thereof, an analog thereof, or a combination thereof. The nonliver disease can be an autoimmune disease, a cardio-/cerebrovascular disease (e.g., stroke, myocardial infarction, and the like), or a neurodegenerative disease, for example.
The present invention also provides a method of reducing expression of c-myc in a cell, the method comprising contacting the cell with an effective amount of ursodeoxycholic acid, salts thereof, or analogs thereof.
Yet another method of involves increasing levels of Bcl-XL in a cell, the method comprising contacting the cell with an effective amount of ursodeoxycholic acid, salts thereof, or analogs thereof.
The present invention also provides a method of inhibiting Bax translocation from the cytoplasm of a cell to a mitochondrial membrane. This is believed to result in the inhibition of changes in the mitochondrion. The method includes a step of contacting the cell with an effective amount of ursodeoxycholic acid, a salt thereof, an analog thereof, or a combination thereof.
A further aspect of the present invention provides a method for limiting apoptosis of a mammalian cell population, the method comprising contacting the cell population with an effective amount of an apoptotic limiting compound selected from the group of ursodeoxycholic acid, a salt thereof, an analog thereof, and a combination thereof, wherein the apoptosis is induced by a membrane damaging agent selected from the group consisting of unconjugated bilirubin, conjugated bilirubin, and a combination thereof.
As mentioned above, the cell population can be hepatocytes, astrocytes, and the like. The contacting step can occur in vitro, in vivo, and a combination thereof. In one embodiment, the cell population is a human cell population.
Preferably, the step of contacting comprises administering to a patient an effective amount of an apoptotic limiting compound selected from the group of ursodeoxycholic acid, a salt thereof, an analog thereof, and a combination thereof In accordance with the present invention, the apoptotic limiting compound can be administered in combination with a pharmaceutically acceptable carrier. Alternatively, administering the apoptotic limiting compound can be administered parenterally. In another embodiment, administering the apoptotic limiting compound can be administered orally.